Compatibility of eugenol anesthesia with classroom physiology experiments on nerve and muscle of frogs

Frogs serve as valuable model organisms for studying physiological responses of nerve, skeletal muscle, and the heart in undergraduate biology labs. Induced hypothermia before pithing is the traditional method of euthanasia, but some veterinary guidelines discourage this practice and recommend chemical anesthesia instead. However, the most commonly used anesthetic, MS-222, may negatively interfere with physiology experiments. We sought an alternative anesthetic and tested the hypothesis that eugenol anesthesia is compatible with classroom nerve and muscle experiments on frogs. Bullfrogs were euthanized after eugenol anesthesia or chilling. Additional groups of frogs were soaked in eugenol for extended durations of 30, 60, or 90 minutes beyond reflex loss. The sciatic nerve was stimulated while the gastrocnemius force was measured using an isometric force transducer. The threshold voltage eliciting a muscle contraction and the peak isometric twitch force were recorded at 15-minute intervals for 2 hours. Eugenol reliably induced loss of reflexes in approximately 20 to 35 minutes. While eugenol increased the threshold voltage required for a muscle contraction and decreased force production, neuromuscular preparations maintained the ability to respond adequately to electrical stimulation, supporting eugenol's viability as an alternative anesthetic for classroom experiments. To further characterize the anesthetic's effects, isolated sciatic nerves were soaked in eugenol solutions and evaluated for compound action potential amplitude and action potential threshold voltage. Eugenol reduced maximum compound action potential amplitude and increased the action potential threshold voltage, with inhibitory effects dependent on the duration of exposure and reversible upon soaking in Ringer's solution.<b>NEW & NOTEWORTHY</b> Prior studies, including Medler in 2019 (<i>Adv Physiol Educ</i> 43: 69-75, 2019), have shown that the primary anesthetic used in fish and other aquatic vertebrates, MS-222, blocks nerve and muscle function and thus interferes with classroom neuromuscular physiology demonstrations in frogs. In this study, we show that eugenol anesthesia is practical and effective for classroom experiments, and we characterize the effect of eugenol on neuromuscular physiology over different anesthetic durations.